DESCRIPTION (from applicant's abstract): The human norepinephrine transporter (hNET) is a high affinity binding site for many psychotherapeutic compounds, including those with antidepressant efficacy (e.g. tricyclic antidepressants). The uptake of norepinephrine (NE) by the NET is the principal mechanism by which the action of NE is terminated at the noradrenergic synapse. Regulation of NET activity in the plasma membrane, therefore, represents an important candidate mechanism through which modulation of noradrenergic ransmission can occur. Despite the fact that many psychoactive compounds bind to the NET, the regulation of NET function induced by these ligands is poorly understood. Our preliminary data demonstrate that certain inhibitors (those which are antidepressants) of NET down-regulate NET function. In fact, exposure to certain NET inhibitors reduces NET function in he absence of the inhibitor for a period of time greater than the initial exposure period. The implication of this data is that occupation-induced down-regulation of NET function may contribute to the therapeutic/ pharmacological action of drugs that bind to the NET. The goals of this proposal are to examine the ability of NET ligands to induce NET down-regulation in in vitro and in vivo preparations, and to elucidate the molecular mechanisms responsible for ligand-induced NET down-regulation. Following continuous exposure of intact NET-expressing cells to NET ligands, studies will examine: (1) NET uptake capacity (function) in in vitro uptake assays, (2) the possibility that there is a rapid redistribution of NET from the plasma membrane surface,(3) the role of protein kinases in ligand-induced NET regulation, and (4) the turnover of NET protein and levels of NET messenger RNA. The biological relevance of NET ligand-induced regulation of NET in vitro will be established by studying NET function in brain slices following treatment of rats with NET ligands. Emphasis in in vitro and in vivo studies will be placed on the temporal aspects of regulation and recovery, because slow recovery from inhibitorinduced down-regulation may imply that antidepressant compounds do not need to be present at the NET in order to inhibit uptake. This information may have a significant impact on treatment regimens of NET inhibitors in the management of psychiatric diseases. Overall, the proposed studies will reveal the basic principles of the relationship between drug exposure and NET regulation and will provide important clues relevant to the pharmacological actions of psychoactive agents that bind to the net.